Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue

A method for the isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and sulfadimethoxine-fortified fish muscle tissue samples (0.5 g) were blended with octadecyisilyl (C18, 40 micrograms, 18% load, endcapped) derivatized silica packing material. A column made from the C18/fish tissue blend was first washed with hexane (8 mL), following which the sulfadimethoxine was eluted with dichloromethane (8 mL). The eluant contained sulfadimethoxine analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 270 nm). Standard curves for sulfadimethoxine isolated from fortified samples were linear (0.999 +/- 0.001) with an average relative percentage recovery of 101.1 +/- 4.2% for the concentration range (50, 100, 200, 400, 800, and 1600 ng/g) examined using sulfamethoxazole as the internal standard. The interassay variability was 10.7 +/- 8.2% with an intra-assay variability of 2.2%.     

Molecularly imprinted polymer online solid-phase extraction coupled with high-performance liquid chromatography-UV for the determination of three sulfonamides in pork and chicken

A selective imprinted amino-functionalized silica gel sorbent was prepared by combining a surface molecular imprinting technique with a sol-gel process for online solid-phase extraction-HPLC determination of three trace sulfonamides in pork and chicken muscle. The imprinted functionalized silica gel sorbent exhibited selectivity and fast kinetics for the adsorption and desorption of sulfonamides. With a sample loading flow rate of 4 mL min (-1) for 12.5 min, enhancement factors and detection limits for three sulfonamides ( S/ N = 3) were achieved. The precision (RSD) for nine replicate online sorbent extractions of 5 microg L (-1) sulfonamides was less than 4.5%. The sorbent also offered good linearity ( r (2) > 0.99) for online solid-phase extraction of trace levels of sulfonamides. The method was applied to the determination of sulfonamides in pork and chicken muscle samples. The prepared polymer sorbent shows promise for online solid-phase extraction for HPLC determination of trace levels of sulfonamides in pork and chicken samples.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Development of an enzyme-linked immunosorbent assay for seven sulfonamide residues and investigation of matrix effects from different food samples

Direct competitive enzyme-linked immunosorbent assays (ELISA) were developed to detect a broad range of sulfonamides in various matrices. Screening for this class of antibiotics in pig muscle, chicken muscle, fish, and egg extracts was accomplished by simple, rapid extraction methods carried out with only phosphate-buffered saline (PBS) buffer. Twenty milliliters of extract solution was added to 4 g of sample to extract the sulfonamide residues, and sample extracts diluted with assay buffer were directly analyzed by ELISA; matrix effects could be avoided with 1:5 dilution of pig muscle, chicken muscle, and egg extracts with PBS and 1:5 dilution of fish extract with 1% bovine serum albumin (BSA)-PBS. For liver sample, the extraction method was a little more complicated; 2 g of sample was added to 20 mL of ethanol, mixed, and then centrifuged. The solvent of 10 mL of the upper liquid was removed, and the residues were dissolved in 10 mL of PBS and then filtered; the filtrate was diluted two-fold with 0.5% BSA-PBS for ELISA. These common methods were able to detect seven sulfonamide residues such as sulfisozole, sulfathiazole, sufameter, sulfamethoxypyridazine, sulfapyridine, sulfamethizole, and sulfachlorpyridazine in pig muscle, liver, chicken muscle, egg, and fish. The assay's detection limits for these compounds were less than 100 microg kg-1. Various extraction methods were tested, and the average recovery (n=3) of 100 microg kg-1 for the matrices was found to range from 77.3 to 123.7%.     

Development of an immunochromatography strip for the rapid detection of 12 fluoroquinolones in chicken muscle and liver

A rapid and sensitive colloidal gold immunochromatography test strip based on one monoclonal antibody with broad-specificity, which can detect 12 fluoroquinolones (FQs), was developed. Antigen and goat anti-mouse IgG were respectively drawn on NC membrane as test line and control line. Gold-labeled antibody was added on a pad and put on one end of the membrane. Fluoroquinolones in sample solution compete with antigen combined on NC membrane for the gold-labeled antibody. When enough fluoroquinolone exists, the test line vanishes as there are no spare gold-labeled antibodies that can bind with antigen on the membrane. The control line always exists when the antibody is activated. The lowest detection limits of the FQs in spiked chicken muscle and chicken liver samples were 25 ng mL(-1) for norfloxacin and pefloxacin. The lowest detection limit for the other 10 FQs (enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, lomefloxacin, enoxacin, danofloxacin, amifloxacin, oxolinic acid, and marbofloxacin) was 50 ng mL(-1). The whole process involving sample preparation and detection can be finished in <10 min. The results demonstrate that the developed method can be potentially used as a screening tool for the determination of 12 FQ residues in a large amount of samples on site.     

Immunochromatography using colloidal gold-antibody probe for the detection of atrazine in water samples

An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.     

Quantitative thin layer chromatographic multi-sulfonamide screening procedure: collaborative study

A thin layer chromatographic procedure suitable for detection of multiple sulfonamides at 0.1 ppm was studied in an interlaboratory collaborative study. Sulfamethazine, sulfadimethoxine, and sulfaquinoxaline were variously analyzed in liver and muscle tissues from swine, turkey, and duck. The average recovery for all drugs across all tissues was 95%. The corresponding repeatability and reproducibility were 7.7% and 10.5%, respectively.     

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycin in Swine tissues

A monoclonal antibody (Mab) was produced by using the kanamycin-glutaraldehyde-bovine serum albumin (Kan-GDA-BSA) conjugate as the immunogen. The anti-Kan Mab exhibited high cross-reactivity with tobramycin (Tob) and slight or negligible cross-reactivity with other aminoglycosides. The specificity and cross-reactivity of this Mab are discussed regarding the three-dimensional, computer-generated molecular models of the aminoglycosides. Using this Mab, a rapid enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based strip test for Kan and Tob were developed. The rapid ELISA showed a 50% inhibition value (IC 50) of 0.83 ng/mL for Kan and 0.89 ng/mL for Tob with the analysis time less than 40 min, and the recoveries from spiked swine tissues at levels of 25-200 microg/kg ranged from 52% to 96% for Kan and 61% to 86% for Tob. In contrast, the strip test for Kan or Tob had a visual detection limit of 5 ng/mL in PBS, 50 microg/kg in meat or liver, and 100 microg/kg in kidney, and the results can be judged within 5-10 min. Observed positive samples judged by the strip test can be further quantitated by ELISA, hence the two assays can complement each other for rapid detection of residual Kan and Tob in swine tissues. Moreover, physical-chemical factors that affected the ELISA and strip test performance were also investigated. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was determined followed by a theoretical explanation of the effects.     

固相萃取-高效液相色谱串联质谱法同时测定有机肥料中13种抗生素残留

建立了同时测定有机肥料中磺胺类、四环素类和喹诺酮类13种抗生素残留的高效液相色谱-串联质谱法,并对样品的前处理方法、仪器分析参数进行了优化。结果表明,13种抗生素在50~1 000 ng/mL间线性关系良好,相关系数均达到0.999以上,检出限在0.02~0.04 mg/kg,样品加标回收率在69%~111%之间,具有良好的重现性。该方法具有灵敏度高、准确度高等优点,可用于有机肥料中13种抗生素残留的同时检测。运用该方法在有机肥料实际检测中发现恩诺沙星、诺氟沙星、土霉素等抗生素残留。     

Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.     

Chloramphenicol extraction from honey, milk, and eggs using polymer monolith microextraction followed by liquid chromatography-mass spectrometry determination

A rapid confirmatory method for monitoring chloramphenicol (CAP) residues in honey, whole milk, and eggs is presented. This method is based on the polymer monolith microextraction (PMME) technique and high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (MS). A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium. To obtain optimum extraction efficiency, several parameters related to PMME were investigated. After dissolution in 20 mM phosphate solution at pH 4.0 and centrifugation, honey, eggs, or milk samples were directly passed through the extraction tube. The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. The eluates were analyzed by LC-MS in the negative-ion mode and by monitoring a pair of isotopic ions for the target compound. The in-source collision-induced dissociation process produced confirmatory ions. The recoveries of CAP from real samples spiked at 0.1-10 ng/g (honey), 0.2-10 ng/mL (milk), and 0.2-10 ng/g (egg) were in the range of 85-102%, with relative standard deviations ranging between 2.1% and 8.9%. The limits of detection (S/N = 3) were 0.02 ng/g, 0.04 ng/mL, and 0.04 ng/g in honey, milk, and eggs, respectively. The proposed method was proved to be robust in monitoring CAP residue in honey, milk, and eggs.     

Simultaneous liquid chromatographic determination of residual synthetic antibacterials in cultured fish

A liquid chromatographic method is described to determine simultaneously the following 11 synthetic antibacterial agents used in a fishery: nitrofuran derivatives furazolidone, nifurpirinol, difurazone, and furamizole; sulfa drugs sulfamerazine, sulfisozole, sulfamonomethoxine, and sulfadimethoxine; and, oxolinic, nalidixic, and piromidic acids. A Nucleosil C18 column was used with tetrahydrofuran-acetonitrile-phosphoric acid-water (29 + 1 + 0.06 + 69.94) as the mobile phase. Pretreatment of the fish meat sample with acetone extraction and alumina column cleanup gave good separation of the LC peaks without interference from any other components. Recovery of the antibacterial agents was ca 80%. The lower limit of detection of the drugs was 1-2 ng for 10 microL injection.     

Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of olaquindox residues

A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 μg/kg and 1.70 ± 0.26 μg/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 μg/kg and 0.31 ± 0.07 μg/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 μg/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.     

Residue depletion of nitrovin in chicken after oral administration

In this study, the residue depletion of nitrovin in chicken was studied after feeding the birds with dietary feeds containing 10 mg/kg of nitrovin for 7 consecutive days. Tissues (muscle, fat, kidney, and liver) and plasma were collected at different withdrawal periods and determined by a high-performance liquid chromatography-ultraviolet (HPLC-UV) method. The limit of detection for nitrovin in tissue and plasma samples was 0.1 ng/(g or mL), and the inter- and intrarecoveries from the blank fortified samples were in the range of 71.1-85.7%. At the withdrawal period of 0 days, the residue concentration of nitrovin in plasma was the highest (average of 84.98 ng/mL) compared to those in muscle, fat, liver, and kidney (average of 21.04, 61.18, 24.04, and 68.28 ng/g, respectively). At the withdrawal period of 28 days, the residue levels of nitrovin in muscle, fat, liver, and plasma were all higher than 1.0 ng/(g or mL) and the highest concentration was in liver (average of 5.8 ng/g). These data are in support of the ban of nitrovin as a feed additive in food-producing animals.     

Rapid and sensitive determination of sulfonamide residues in milk and chicken muscle by microfluidic chip electrophoresis

A new, rapid, and sensitive method was proposed for the determination of sulfonamide residues in milk and chicken muscle samples by microchip electrophoresis with laser-induced fluorescence detection. Separation of fluorescamine-labeled sulfonamides was accomplished by using a buffer containing 5 mmol/L boric acid and 1% (w/v) polyvinyl alcohol (PVA). The pH, amount of PVA, and concentration of boric acid in the running buffer were found to have great influence on the separation. By optimizing these conditions, the separation of four sulfonamides, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide, was achieved within 1 min with limits of detection (S/N = 3) of 0.2-2.3 μg/L, which are well below the maximum residue limit. The proposed method also exhibited very good repeatability; the relative standard deviations for both within-day and between-day measurements were ≤3.0%. With a simplified sample pretreatment protocol, fast determination of sulfonamides in real samples was successfully performed with standard addition recoveries of 93.3-100.8 and 82.9-92.3%, respectively.     

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.     

Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of furazolidone in pork muscle tissue

A method for the isolation and liquid chromatographic (LC) determination of furazolidone in pork muscle tissue is presented. Blank or furazolidone-fortified pork muscle tissue samples (0.5 g) were blended with octadecylsilyl (C18, 18% load, endcapped, 2 g) derivatized silica. A column made from C18/pork matrix was first washed with hexane (8 mL), followed by elution of furazolidone with ethyl acetate. The ethyl acetate extract was then passed through an activated alumina column. The eluate contained furazolidone that was free from interfering compounds when analyzed by LC with UV detection (photodiode array, 365 nm). Detector response with increasing concentrations of furazolidone isolated from fortified samples was linear (r = 0.998 +/- 0.002) with an average percentage recovery of 89.5 +/- 8.1% for the concentration range (7.8-250 ng/g) examined and resulted in a minimum detectable limit of 390 pg on column, and a detector response of more than 5 times baseline noise. The inter-assay variability was 9.9 +/- 5.4% with an intra-assay variability of 1.5%.     

Development of a chemiluminescent ELISA for determining chloramphenicol in chicken muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.     

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.